Non-invasive in vivo imaging and methods for treating diabetes

ABSTRACT

The present invention provides novel drug discovery platforms and methods for treating diabetes.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/265,212 filed Sep. 14, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/515,000 filed Oct. 15, 2014, now U.S. Pat. No.9,463,205 issued Oct. 11, 2016, which is a divisional of U.S.application Ser. No. 12/199,473 filed Aug. 27, 2008, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/969,437filed Aug. 31, 2007, 61/042,482 filed Apr. 4, 2008, and 60/989,038 filedNov. 19, 2007, all incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Fundamental understanding of cellular processes in health and diseasehas been gained by studying cells of various tissues in vitro. However,results obtained from experiments in vitro are often not sufficient toexplain the performance of cells in more physiological settings likewhole organs or living organisms. To place observations made in an invitro system into a physiological context, studies have to be performedunder in vivo conditions. In recent years an increasing number ofapproaches have been made to investigate cell function in situ utilizingimaging techniques. Unfortunately, non-invasive imaging techniques likecomputer tomography (CT), magnet resonance imaging (MRI), positronemission tomography (PET) or bioluminescence imaging (BLI) lack cellularresolution¹. On the other hand, confocal and two-photon laser-scanningmicroscopy (LSM) provide sub-cellular resolution but have a fairlylimited working distance and imaging depth². Accessing target cells forthe application of LSM is mostly invasive and often excludes thepossibility of repetitive examinations.

SUMMARY OF THE INVENTION

The present invention provides methods for drug development comprising:

(a) engrafting target cells into the eye of a test animal, wherein oneor more cellular component of therapeutic interest in the transplantedtarget cells are fluorescently labeled;

(b) contacting the target cells with one or more test compounds; and

(c) performing non-invasive fluorescent imaging on the eye of the testanimal, wherein the fluorescent imaging is used to detect testcompound-induced changes in one or more of activity, location, andamount of the fluorescently labeled cellular components of therapeuticinterest in the engrafted target cells, wherein the changes identifythose test compounds that may provide a therapeutic benefit to thetarget cells.

In another aspect, the present invention provides methods for treating asubject with type I diabetes, comprising transplanting into the eye of asubject with type I diabetes an amount effective of an insulin-producingcell to promote insulin production in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Pancreatic islets transplanted into the anterior chamber of theeye.

(a) Islet transplantation into the anterior chamber of the eye. (b) Setup for non-invasive in vivo imaging. (c) Digital photograph of isletsengrafted on the iris in the anterior chamber of the eye. (d-g) Eyesections containing islet grafts showing insulin-immunoreactive β-cells(red) and glucagon immunoreactive β-cells (green) at differenttime-points after transplantation.(h) The ratio of insulin to glucagon immunoreactive cells in islets inthe pancreas and in the anterior chamber of the eye at indicatedtime-points after transplantation. (i) Plasma glucose levels during anintraperitoneal glucose tolerance test in streptozotocin-treated micetransplanted with islets into the eye (n=8, black) and under the kidneycapsule (n=2, red).

FIG. 2. Non-invasive imaging of islet engraftment and vascularization.Images of an individual RIP-GFP islet graft 4 months aftertransplantation (optical section captured at a depth of 51 μm). (a)β-cell GFP fluorescence (green), (b) intravenously injected Texas Redfluorescence (red), and (c-d) 2D projection of an image stackcorresponding to 100 μm thickness. (e-p) Image projections (110 μmthick) of an individual RIP-GFP islet graft at day 3, 7, 14 and 28 aftertransplantation. GFP fluorescence of β-cells (e,h,k,n) and Texas Redfluorescence in blood vessels (f,i,l,o) are displayed separately and asan overlay (g,j,m,p). Scale bars, 100 μm. (q-r) Quantification ofresults shown in (e-p) The number of analyzed islet grafts is indicatedin brackets at respective time-points.

FIG. 3. In vivo imaging of β-cell function. (a-e) Fluorescence images ofFluo-4 and Fura-Red at indicated time points after systemic applicationof glibenclamide (1 mg/kg) at 3 min. (f) Whole frame Fluo-4/Fura-Redratio change in response to given glibenclamide stimulus, start ofstimulation indicated by arrow. (g) Ratio change in individual cellsthroughout the islet as indicated in panel (a). (h-i) Maximumprojections of Fluo-4 (green) and Fura-Red (red) fluorescence of a wholeislet before (h) and after stimulation with glibenclamide (i). (j-k)Ratiometric display of Fluo-4/Fura-Red of the islet in panel (h) and(i). Scale bar, 50 μm.

FIG. 4. Non-invasive in vivo imaging of β-cell death. (a-h) Imageprojections of an individual RIP-GFP islet graft in the anterior chamberof the eye. Under normal conditions, (a) β-cell GFP fluorescence, (b)reflection from the endocrine cells and (c) undetectable annexin V-APClabelling of the graft. (d) Overlay image of (a-c) with the reflectionimage in blue. Twenty-four hours after induction of β-cell death, (e)β-cell GFP fluorescence, (f) reflection and (g) strong annexin V-APCfluorescence of the islet graft. (h) Overlay image of (e-g) with thereflection image in blue. (i-j) High magnification images of an isletgraft area strongly labelled with annexin V-APC after induction ofβ-cell death. (i) β-cell GFP fluorescence, (j) annexin V-APCfluorescence and (k) overlay of (i-j). (l) Overlay of (k) with thereflection image (blue). Scale bars, 100 μm.

FIG. 5. Non-invasive imaging of islet engraftment and vascularization.(a-f) Image projections (110 μm thick) of the same RIP-GFP islet graftas displayed in FIG. 2 are shown at 2 and 4 months aftertransplantation. GFP fluorescence of β-cells (a,d) and Texas Redfluorescence in blood vessels (b,e) are displayed separately and as anoverlay (c,f). Scale bar, 100 μm.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides methods for drugdevelopment comprising:

(a) engrafting target cells into the eye of a test animal, wherein oneor more cellular component of therapeutic interest in the transplantedtarget cells are fluorescently labeled;

(b) contacting the target cells with one or more test compounds; and

(c) performing non-invasive fluorescent imaging on the eye of the testanimal, wherein the fluorescent imaging is used to detect testcompound-induced changes in one or more of activity, location, andamount of the fluorescently labeled cellular components of therapeuticinterest in the engrafted target cells, wherein the changes identifythose test compounds that may provide a therapeutic benefit to thetarget cells.

As used herein, the target cells to be transplanted may be individualcells, a plurality of cells of the same type, or a plurality ofdifferent cell types, such as tissues/tissue portions. The cells may beof any type desired to be assessed, including but not limited toendocrine cells (including but not limited to pancreatic beta cells),and cells derived from any tissue type, including but not limited tofat, muscle, brain, liver, kidney, heart, and lungs.

The methods of the invention provide a novel platform for non-invasivein vivo drug development studies that take into account the physiologyand pathophysiology of any cell or tissue. In one embodiment, theanterior chamber of the eye can be used as a versatile natural bodywindow to clarify, for the first time, the integration of complexsignaling networks at the cellular level under in vivo conditions.Employing the anterior chamber of the eye as an in vivo model forassessing activity of one or more cellular components enables thecontinuous monitoring of, for example, morphology, vascularization,innervation, cell death, cell proliferation, cell development (includingbut not limited to stem cell development, tumor cell development, etc.),gene expression and cell signaling. The use of this platform can beused, for example, to elucidate the effects of modulatory inputs from,for example, the hormonal and neuronal system, as well as fromautocrine/paracrine signals of endocrine or vascular cells. Furthermore,it serves as a novel approach for non-invasive in vivo studies ofcell/tissue function and survival under healthy and non-healthyconditions. Thus, the model system is ideal for use, for example, indrug discovery and testing of drug candidates (including but mot limitedto drug candidates for treating cancer, diabetes, etc.) in vivo.

The test mammal can be any suitable test mammal in which cells can betransplanted into the anterior chamber of the eye, including but notlimited to mice, monkeys, rabbits, dogs, rats, and pigs.

The anterior chamber of the eye comprises the front portion of the eye,and includes the structure in front of the vitreous humour, as well asthe cornea, iris, ciliary body, and lens. Transplantation of targetcells into the anterior chamber of the eye can comprise placement of thecells into any one or more of these anterior eye chamber compartments,so long as the target cells can be observed and fluorescent signals fromthe cells can be non-invasively visualized. In one non-limiting example,target cells are transplanted via injection through the cornea, allowingengraftment of the transplanted target cells onto the iris, permittingobservation and imaging through the cornea.

The one or more cellular components of interest in the transplanted testcells are fluorescently labeled. The labeling may be direct (ie:covalent interaction) or indirect (non-covalent interaction) cells maybe labeled before or after transplantation. Pre-transplantation labelingcan be accomplished by any means known in the art, including but notlimited to recombinant DNA techniques, such as transfection of the cellswith an expression construct that will express a fluorescentprotein-labeled cellular component of interest. Any fluorescent proteincan be used including but not limited to green fluorescent protein andall of its variants. Post-transplantation labeling can also be by anymeans known to those of skill in the art, including but not limited toinjection of fluorescent stains of interest and contact with labeledantibodies.

Fluorescence imaging on the anterior eye chamber can be accomplished byany technique known to those of skill in the art, including but notlimited to laser scanning microscopy. In one embodiment, the methodsinvolve stimulating fluorescence from the labeled cellular components ofinterest by laser stimulation at appropriate wavelength(s) tonon-invasively obtain fluorescence images of the cellular components inthe transplanted cells.

The activity of any cellular component of interest can be assessed viathe methods of the present invention. Such activity can be anycharacteristic of the cellular component of interest that can beassessed based on detection of fluorescent signals from the cellularcomponent of interest, including but not limited to expression,distribution, localization, amount, kinetics/dynamic changes,modifications, and oscillations. In a further embodiment, the methodscomprise assessing activity of the cellular component of interest overtime. In this embodiment, the methods comprise performing fluorescentimaging at multiple time points, and changes in activity of the cellularcomponent of interest can be assessed. In an embodiment of all of theembodiments herein, the assessment is done within individual cells.

In one non-limiting embodiment, the cellular component of interestcomprises a component of a signal transduction pathway, and the methodscomprise assessing activity of the signal transduction pathway byassessing activity of the one or more cellular components of interestover time. In a further non-limiting embodiment, the test cells comprisepancreatic beta cells.

The methods comprise contacting the target cells with one or more testcompounds and assessing the activity of the fluorescently labeledcellular components of interest in the target cells in response to theone or more test compounds. Such contacting of the transplanted cellswith the one or more test compounds can be pre or post-transplantation;preferably post-transplantation.

In the examples below, we transplanted isolated pancreatic islets ofLangerhans into the anterior chamber of the eye. Islets of Langerhansare composed of several different cell types, including alpha-, beta-and delta-cells. These clusters of cells represent the endocrinepancreas and are of major importance for glucose homeostasis.Insufficient release of insulin from beta-cells in response to elevatedblood glucose levels, leads to diabetes. The regulation of glucoseinduced insulin secretion from beta-cells is a complex process,modulated by autocrine, paracrine, hormonal and neuronal factors.Therefore, studies on vascularized and innervated islets of Langerhansare necessary to understand the mechanisms leading to hormone secretionin health and disease. However, due to the scattered distribution ofislets of Langerhans throughout the exocrine pancreas and the anatomy ofthe rodent pancreas in particular, non-invasive longitudinal in vivostudies of the islets of Langerhans at single-cell resolution arecurrently not feasible.

We show below, that after transplantation to the anterior chamber of theeye, isolated islets readily engrafted. Diabetic mice could be renderednormoglycemic by transplanting islets of Langerhans to the anteriorchamber of the eye and these mice showed identical responses to glucosetolerance tests compared to control mice. We were able to longitudinallymonitor morphology of the islets and follow the revascularizationprocess. We could also repetitively measure systemically induced changesin cytoplasmic free Ca²⁺ concentration in beta-cells of the same islet.Finally, we non-invasively monitor chemically induced cell death inislets after systemic injection of a beta-cell toxin.

This platform enables non-invasive assessment of multiple morphologicaland functional parameters in vascularized and innervated tissue.Although the examples focus on pancreatic islets of Langerhans theintroduced platform can be used to investigate all kinds of tissues.Different types of tissues, transplanted to the anterior chamber of theeye, have been shown to attract vessels and nerves and establishorganotypic vascularization^(10,12) and innervation^(4,5,13). Thisallows studying tissues in a setting comparable to their naturalsurrounding without having to access the tissue in an invasive manner.Furthermore, due to the characteristics of the eye, the target cells andtheir regulatory input can be modulated not only systemically but alsolocally without difficulty. Substances can be applied topically onto theeye or injected into the anterior chamber. Additionally, perfusion ofthe anterior chamber repetitively allows exchange of the aqueous humoror loading of the graft with fluorescent indicators.

When utilizing the anterior chamber of the eye as a transplantationsite, the special local features expressed to optimize visualization andto make the anterior chamber an immune privileged site are taken intoconsideration. Differences in the composition of the aqueous humorinside the anterior chamber and the blood plasma are kept in mind wheninterpreting observations made in the anterior chamber. However, ourstudies have shown no obvious effects on normal graft function by thelocal environment. One possible explanation might be that the inducedneovascularization leads to an adjustment of aqueous humor and bloodplasma composition. Thus, we have demonstrated that the anterior chamberof the eye enables studies of complex biological interactions in an invivo system at single-cell resolution. Its current form is furtherextendible and implementation of newly developed fluorescent proteins,biosensors and transgenic animals will help to investigate numerousparameters important for development, function and survival under bothphysiological and pathophysiological conditions.

In another aspect, the present invention provides methods for treating asubject with type I diabetes, comprising transplanting into the eye of asubject with type I diabetes an amount effective of an insulin-producingcell to promote insulin production in the subject.

As used in this aspect, an “insulin-producing cell” is any cell typethat is capable of producing insulin after transplantation into asubject's eye. Such insulin-producing cells include, but are not limitedto, pancreatic β cells, stem cells, and recombinant cells engineered torelease insulin.

As used herein, “pancreatic β cells” are any population of cells thatcontains pancreatic β islet cells. Such pancreatic β islet cellpopulations include the pancreas, isolated pancreatic islets ofLangerhans (“pancreatic islets”) and isolated pancreatic β islet cells.Methods for pancreatic isolation are well known in the art, and methodsfor isolating pancreatic islets, can be found, for example, in Cejvan etal., Diabetes 52:1176-1181 (2003); Zambre et al., Biochem. Pharmacol.57:1159-1164 (1999), and Fagan et al., Surgery 124:254-259 (1998), andreferences cited therein. Once implanted in the host eye, the beta cellsin these islets begin to make and release insulin

As disclosed herein, the inventors have disclosed a new method formonitoring real-time events in cells such as pancreatic β cells bytransplanting cells into the anterior chamber of the eye. In conductingthese studies, the inventors have determined that transplantation ofpancreatic β cells into the eye has not led to any serious complication,such as vision loss or excessive eye irritation. Thus, given that theeye provides a somewhat immunopriviliged compartment, it is believedthat transplantation of insulin-producing cells into the eye of patientswith type I diabetes can provide the necessary insulin production, whileallowing a lowered dosage of immunosuppressant drugs to be used,reducing the potentially severe side effects of immunosuppression. It isalso believed that transplantation into the eye will reduce any periodof anoxia during transplantation, resulting in an increased survival ofthe transplanted cells.

Transplantation into the eye preferably involves transplantation intothe anterior chamber of the eye. The anterior chamber of the eyecomprises the front portion of the eye, and includes the structure infront of the vitreous humour, as well as the cornea, iris, ciliary body,and lens. Transplantation of the insulin-producing cells into theanterior chamber of the eye can comprise placement of the cells into anyone or more of these anterior eye chamber compartments. In onenon-limiting example, test cells are transplanted via injection throughthe cornea, allowing engraftment of the transplanted cells onto theiris, permitting observation and imaging through the cornea. Insulinproducing cells, such as pancreatic beta islets, transplanted into theanterior chamber of the eye engrafted on the iris, became vascularized,retain their cellular composition, and respond to stimulation.Furthermore, they can be monitored by non-invasive laser scanningmicroscopy (LSM) allowed in vivo imaging of islet vascularization, aswell as beta-cell function and insulin release. In these embodiments,the insulin-producing cells or components thereof can be fluorescentlylabeled, and fluorescence imaging can be used to monitor cell activity.

Fluorescence imaging on the anterior eye chamber can be accomplished byany technique known to those of skill in the art, including but notlimited to laser scanning microscopy. In one embodiment, the methodsinvolve stimulating fluorescence from the labeled cellular components ofinterest by laser stimulation at appropriate wavelength(s) tonon-invasively obtain fluorescence images of the cellular components inthe transplanted cells.

Example 1

Impaired insulin release and thereby defect glucose homeostasis in thebody is a hallmark of diabetes (1). Under physiological conditions,insulin release from the pancreatic β-cell is regulated by the complexand concerted actions of cell metabolic activity, autocrine/paracrinesignalling, and continuous input from hormones and neurotransmitters(2). The β-cells, together with other endocrine cell types, are situatedwithin the endocrine pancreas, the islets of Langerhans, which aredensely vascularised (3) and abundantly innervated (4). Therefore, tofully understand the complexity of β-cell signal-transduction and themechanisms controlling insulin release in health and disease, studiesneed to be conducted in vascularised and innervated islets in vivo.Here, we introduce a novel noninvasive technical platform for in vivofluorescence imaging of pancreatic islets transplanted into the anteriorchamber of the mouse eye. Islets transplanted into the anterior chamberof the eye engrafted on the iris, became vascularised, retained cellularcomposition and responded to stimulation. Non-invasive laserscanningmicroscopy (LSM) allowed in vivo imaging of islet vascularization, aswell as β-cell function and death. Our results thus establish the basisfor repetitive non-invasive in vivo investigations of β-cellsignal-transduction, which can be performed longitudinally under bothnormal and diabetic conditions.

Methods and Materials Mouse Models.

C57BL6 and Tie2-GFP mice (STOCK Tg(TIE2GFP)287Sato/J) were purchasedfrom the Jackson Laboratories (Bar Harbor, Me.). RIP-GFP mice weregenerated at a core-facility at Karolinska Institutet and werecharacterized by a normal glucose tolerance and β-cell restrictedexpression of GFP (see Methods). All experiments were approved by thelocal animal ethics committees at Karolinska Institutet and theUniversity of Miami.

Transplantation of Pancreatic Islets to the Anterior Chamber of the Eye.

Pancreatic islets were isolated and cultured as described (25). Thirtyto three hundred islets were transferred from culture media to sterilePBS and aspirated into a 27G eye cannula connected to a 1 ml Hamiltonsyringe (Hamilton, Reno, Nev.) via a 0.4 mm polythene tubing (PortexLimited, Kent, England). Mice were anesthetized using isoflurane(Isoflurane, Abott Scandinavia AB, Solna, Sweden) and 0.1 ml/kg ofTemgesic (Schering-Plough, NJ) was subcutaneously injected to relievepost-operative pain. Under a stereomicroscope, the cornea was puncturedclose to the sclera at the bottom part of the eye with a 27G needle.Great care was taken not to damage the iris and to avoid bleeding. Next,the blunt eye cannula was gently inserted and the islets were slowlyinjected into the anterior chamber where they settled on the iris. Afterinjection, the cannula was carefully withdrawn and the animal was leftlying on the side before awakening. The mice quickly recovered andshowed no signs of stress or irritation from the transplanted eye.

Intravital Imaging of Islets Transplanted to the Anterior Chamber of theEye.

Previously transplanted mice were anesthetized with a 40% oxygen and ˜2%isoflurane (Isoflurane) mixture, placed on a heating pad. The mouse headwas restrained with a stereotaxic headholder (SG-4N, Narishige, Tokyo,Japan) and positioned with the eye containing the engrafted isletsfacing up. The eyelid was carefully pulled back and the eye was gentlyheld at the corneoscleral junction with a pair of tweezers attached to aUST-2 Solid Universal Joint (Narishige). The tips of the tweezers werecovered with a single piece of polythene tubing creating a loop betweenthe two tips. This arrangement permitted a flexible but stable fixationof the head and eye without causing damage or disrupting the bloodcirculation in the eye. An upright Leica DMLFSA microscope, equippedwith a TCS-SP2-AOBS confocal scanner and lasers for two-photonexcitation25, was used for imaging together with long distancewater-dipping lenses (Leica HXC APO 10×0.3 W, 20×0.5 W, 40×0.8 W), usingfiltered saline as an immersion liquid. For visualization of bloodvessels, Texas Red (100 μl of 10 mg/ml; Molecular Probes, Eugene, Oreg.)was intravenously injected via the tail vein. GFP and Texas Red wereexcited at 890 nm and emission light was collected and separated ontotwo nondescanned detectors using a dicronic mirror (RSP560) and emissionfilters (BP 525/50 and BP 640/20). The images captured with TPLSM weredenoised using wavelet filtering as previously described (27). Forvisualization of cell death, 100 μl of annexin V-APC (Molecular Probes)were intravenously injected via the tail vein. GFP was excited at 488 nm(35% AOTF) and emission light was collected between 495-530 nm.Reflected light was imaged by illumination at 546 nm (35% AOTF) andcollection between 539-547 nm. APC was excited at 633 nm (75% AOTF),with collection of emission light between 644-680 nm. Initial studiesshowed weak annexin V-APC labelling of RIPGFP islet grafts ˜40 min afteradministration with a gradual increase. The islet grafts were imaged 4-6h after administration of annexin V-APC. The image stacks captured withCLSM were denoised using median filtering. All displayed fluorescenceimages have been subjected to changes in brightness and contrast foroptimal visualization.

Generation of RIP-GFP Mice.

RIP-GFP mice were generated by injections of the RIP1.EGFP expressioncassette (rat insulin-1 promoter −410/+1 bp-EGFPSV40polyA) into one-cellstage embryos from B6CBAF1/Crl donors. The obtained F0 generation wasscored for RIP1.EGFP genomic integration by PCR analysis. The RIP1.EGFPtransgene was observed in seven potential transgenic founders (17.5%),which were mated with inbred C57Bl/6NCrl mice to generate F1 animals.The founder lines were screened with regards to 1) the expression of GFPin β-cells as determined by immunostaining, and 2) animal and cellphysiology. The RIP1.EGFP founder line #29 was found to have a normalglucose tolerance when compared to control animals and β-cell restrictedexpression of GFP, and was selected for homozygote breeding.

Immunohistochemistry.

Mice were killed by exposure to a rising concentration of CO2 followedby cervical dislocation, after 3, 7, 14, and 28 days subsequent tointraocular islet transplantation (n=12). The graft-bearing eyes wereremoved and postfixed for 1 h in 4% paraformaldehyde. Aftercryoprotection by sucrose substitution (10%, 20%, and 30% in PBS),vertical sections of the eyes were cut on a cryostat (14 μm). Sectionswere washed in PBS (3×10 min) and incubated in PBS containing 5% bovineserum albumin and 0.1% triton (1 h). Thereafter, sections were incubatedovernight in PBS with anti insulin (1:500, Accurate Chemical &Scientific Corp., NY), and anti glucagon (1:5000; Sigma, St Louis Mo.).Immunostaining was visualized using either Alexa 488 or Alexa 568conjugated secondary antibodies (1:500; Molecular Probes). Cell nucleiwere stained with DAPI (Molecular Probes). Slides were mounted withVectamount and coverslipped. Serial cross sections of eyes containingislets were examined for the presence of insulin and glucagon with anAxiophot fluorescence microscope (Zeiss, Oberkochen, Germany) and adual-channel laser scanning confocal microscope (Olympus Fluoview,Olympus America Inc., Melville, N.Y.). All immunostaining images weredigitally acquired and recompiled (Photoshop 5.0; Adobe, San Jose,Calif.). Sections were viewed at 10× and 40× magnification. Analyseswere done on digitized fluorescence microscopic images using ZeissAxiovision software. Measured parameters (e.g. ratio insulinimmunoreactive cells/glucagon-immunoreactive cells) were calculated asthe average from at least three adjacent sections from at least twoseparate islets per eye. The results from three eyes were averaged. Onlycells that had a clearly labeled nucleus (DAPI staining) were includedin the analyses. Data are presented as mean±SEM.

Quantification of Islet Graft Vasculature.

The vessel density was determined as the number of vessel segments pergraft area. A vessel segment was defined as a single vessel or a branchof a vessel. The β-cell GFP fluorescence was used to define the graftarea. Two optical sections were quantified from each islet graft. Theoptical sections were selected from image series of z-stacks. The firstsection was selected at the deepest level in the graft, without loss ofsignal. The second section was selected in the middle of the graft,between the surface and the deepest section. The images forquantification were captured using 10× or 20× lenses and a zoom factorof 2.0 or greater. Data are presented as mean±SEM. The quantification ofthe vasculature was made with the Leica Confocal Software (version2.61).

In Vivo Recording of [Ca2+]i Changes.

To assess function of β-cells in vivo, islets transplanted to theanterior chamber of the eye were loaded with a mixture of the Ca2+indicators Fluo-4 and Fura-Red. Applying these two dyes simultaneouslyallowed ratiometric [Ca2+]i measurements with excitation spectra in thevisible wavelengths (29). To achieve loading, the anterior chamber wasperfused with an extracellular solution consisting of (in mM) 140 NaCl,5 KCl, 2 NaHCO₃, 1 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 10 HEPES, 3 glucose(pH 7.4 with KOH), containing 500 μM of each AM ester of Fluo-4 andFura-Red. For perfusion, mice were anesthetized with 10 ml/kg of amixture of 1 part Hypnorm (0.315 mg/ml fentanyl and 10 mg/ml fluanisone,VetaPharma, Leeds, UK), 1 part Dormicum (5 mg/ml, Roche, BaselSwitzerland) and 2 parts sterile water. To prolong anesthesia,subsequent injections of the mixture (4 ml/kg) were administered every30-40 min. Mice were placed on the microscope setup as described above,and body temperature was controlled via a rectal probe connected to thecontrol unit of the heating pad. Micropipettes were pulled fromborosilicate glass capillaries on a horizontal programmable puller (DMZUniversal Puller, Zeitz-Instrumente, Augsburg, Germany) and broken to atip outer diameter of 60-100 μm. Adjacent micropipette tips werebevelled at an angle of 30° using a rotating wheel grinder (model BV10,Sutter Instruments Co., Novato, Calif., USA). Micropipettes wereintroduced into the anterior chamber by penetrating the cornea atshallow angles on opposite sides of the

eye using two micromanipulator units (Eppendorf, Hamburg, Germany). Carewas taken not to damage the iris or islet grafts. One micropipette wasattached to a reservoir filled with extracellular solution viapolyethylene tubing. The height of the reservoir was adjusted to assurea constant intracameral pressure of about 15 mmHg. The secondmicropipette was connected to a 1 ml syringe. Rate and volume of inflowwas controlled by a syringe pump (Univector). Islet cells were loadedwith Ca2+ indicators by perfusing the anterior chamber withextracellular solution containing Fluo-4 and Fura-Red AM esters for 40min at 3 μl/min. Subsequently, the dye containing solution was washedout by perfusion with extracellular solution for 10 min at 5-6 μl/min.Imaging was performed as described above exciting Fluo-4 and Fura-Red at488 nm (25% AOTF) and collecting emission light for Fluo-4 between495-535 nm and for Fura-Red between 600-700 nm. Systemic stimulation ofinsulin release was achieved by intravenous injection of glibenclamide(1 mg/kg) via the tail vein. After finishing imaging, mice weresubcutaneously injected with 0.1 ml/kg of Temgesic (Schering-Plough, NJ)to relieve post-operative pain.

Results

Pancreatic islets are hardly accessible for in vivo monitoring becausethey are deeply embedded and scattered in the exocrine tissue of thepancreas, constituting 1-2% of the pancreatic volume 5. As aconsequence, the majority of today's functional β-cell studies areconducted in vitro on isolated islets/cells. Isolated islets (6) andespecially pancreatic slices (7) allow functional studies of β-cells ina multi-cellular environment. However, these preparations are restrictedto defined end-points and partially lack input from vascular and nervousconnections. There is an immediate need to monitor β-cell function invivo to understand the complex signalling networks involved in theregulation of insulin release under normal conditions, and why these donot function properly in type 2 diabetes. This is also the case in thecontext of clinical islet transplantation, which is emerging as atherapy for type 1 diabetes (8.) To date, monitoring β-cellsignal-transduction after experimental and clinical islettransplantation has not been possible, which has severely hampered boththe characterization of the graft function and the evaluation of newinterventions (9). LSM has been successfully applied for imaging ofmultiple signaling pathways in the β-cell using isolated islets and cellpreparations (6,10). However, intravital applications of LSM for studiesof β-cell physiology have not been reported.

After islet transplantation, islets recruit a new vasculature (11) aswell as nervous Connections (12), and are capable of maintaining glucosehomeostasis via pulsatile insulin release (12,13). We decided totransplant pancreatic islets into the anterior chamber of the eyebecause the cornea acts as a natural body window that allows noninvasiveimaging of engrafted tissue. The anterior chamber of the eye has beenfrequently used as a transplantation site to study a variety of tissuesincluding pancreas because it is immune privileged (14-17). Mouse isletswere transplanted into the anterior chamber of the eye via injectionthrough the cornea (FIG. 1a ). After transplantation, the isletsengrafted on the iris and were readily observed and imaged through thecornea (FIG. 1b-c ). The transplanted islets engrafted either as singleislets or in groups as islet clusters. Immunohistochemical staining ofengrafted islets showed that the proportion of the insulin-containingβ-cells and glucagon-containing β-cells did not change aftertransplantation and that this proportion was similar to that of isletsin the pancreas (FIG. 1d-h ), which is in agreement with earlier

Studies (14,16,17).

In mice that were rendered diabetic with streptozotocin, transplantingislets into the anterior chamber reversed hyperglycaemia. These micefurther showed physiological responses to glucose challenges (FIG. 1i ),demonstrating that islets

engrafted in the anterior chamber of the eye are functional. To allowidentification of β-cells, islets isolated from transgenic miceexpressing the enhanced green fluorescent protein (GFP) under thecontrol of the rat insulin 1 promoter (RIP-GFP) were transplanted intothe anterior chamber of the eye.

By using two-photon LSM (TPLSM) we imaged simultaneously β-cells by GFPand the vasculature following intravenous injection of 100 μl Texas Red70 kDa dextran (Texas Red, 10 mg/ml) and determined that transplantedislets recruited blood vessels from the iris. Non-invasive TPLSM enabledcapturing of optical sections at different depths in the engraftedislets (FIG. 2a,b ), and thereby 3D (3-dimensional) reconstruction ofboth the β-cell and the vascular morphology within the islet grafts(FIG. 2c-d ). To monitor the dynamics of islet engraftment andvascularization, the same RIP-GFP islets were repeatedly imaged at day3, 7, 14 and 28 after transplantation (FIG. 2e-p ). At day 3, thetransplanted islets were attached to the iris and structuralrearrangements of iris vessels in the vicinity of the islets wereobserved. However, only few vessels were found to have grown into theperipheral regions of the islets (FIG. 2e-g ). At day 7, the isletgrafts appeared thinner but wider compared to day 3, indicating that theislets had further attached and spread out onto the iris. An increasednumber of blood vessels were found in the islets and capillary loopswere found to start penetrating the central islet regions (FIG. 2h-j ).While only minor additional changes in islet structure occurred afterday 7, blood vessels continued to grow, and at day 14 they formed amicrovascular network throughout the islet grafts (FIG. 2k-m ). Betweenday 14 and day 28, the vascular network became denser, and at day 28 itwas characterized by highly tortuous and uniformly sized capillaries(FIG. 2n-p ). The vessel density of the transplanted islets continuouslyincreased during revascularization (FIG. 2q ). The diameter of isletgraft vessels was 8.11±0.53 μm at day 28 (n=5, FIG. 2r ), which issimilar to the intra-islet vasculature in the pancreas (18) and at othertransplantation sites (19). Imaging of islet grafts at two and fourmonths after transplantation showed that the morphology of the β-cellmass and the graft vasculature were similar compared to day 28 (FIG. 5).In conclusion, non-invasive TPLSM enabled longitudinal in vivo imagingof β-cells and the vasculature in islets engrafted in the anteriorchamber of the eye.

The assessment of signal-transduction in vivo at the cellular levelwould allow investigation of islet cells under both physiological andpathophysiological conditions, including regulatory and modulatoryinfluences from paracrine, hormonal, and neuronal signals. To monitorcell function in vivo, we studied changes in cytoplasmatic free calciumconcentration ([Ca2+]i) in islets at the single cell level in theanterior chamber of the eye. Changes in [Ca2+]i are key intracellularsignals in islet cells and serve as a reporter of β-cell function2. Weloaded islets with the Ca2+ indicators Fluo-4 and Fura-Red via perfusionof the anterior chamber of the eye with micropipettes. Applying thesetwo dyes simultaneously allowed ratiometric measurements of [Ca2+]ichanges with LSM and correction for movements of the islets duringimaging. Fluo-4 and Fura-Red labelled the outer layer of the isletshomogenously (FIG. 3h-i ). To stimulate cells, we applied thesulfonylurea compound glibenclamide intravenously. This decreased bloodglucose levels, indicating that the administration was effective (datanot shown). Increases in [Ca2+]i in islet cells, as demonstrated byprominent rises in the Fluo-4/Fura-Red fluorescence ratio, startedwithin 30 to 40 seconds after glibenclamide injection into the tail veinof the mouse and remained high throughout the recording (FIG. 30.[Ca2+]i increased simultaneously in different regions of the islets,reflecting a synchronized response of β-cells within an islet afterstimulation 20 (FIG. 3g ). These results demonstrate that it is feasibleto image islet cell function in vivo employing engrafted islets in theanterior chamber of the eye.

β-cell death is a characteristics of type 1 diabetes (21) and isimplicated in the pathology of type 2 diabetes (22). To date, no methodsexist for continuous monitoring of β-cell death in vivo. Annexin V hasbeen used as a reporter of cell death both under experimental andclinical conditions (23) and has been validated as a marker for β-cellapoptosis after systemic administration (24). To investigate thefeasibility of noninvasive imaging of β-cell death, we transplantedRIP-GFP islets into the anterior chamber of the eye and, after completeengraftment and revascularization, we monitored cell death followingintravenously administered annexin V conjugated to allophycocyanin(APC). Using confocal LSM, GFP- and annexin V-APC fluorescence werecaptured simultaneously with reflected light, whereby the latterprovides detailed structural information of endocrine cells (25).Transplanted RIP-GFP islets imaged in mice with regular blood glucoselevels displayed normal morphology (FIG. 4a-b ) and absence of annexinV-APC labelling (FIG. 4c-d ). Annexin VAPC was only found to label a fewcells in 1 out of 10 RIP-GFP islet grafts (data not shown), indicating alow incidence of cell death in islets engrafted in the anterior chamberof the eye. We induced β-cell death in mice transplanted with RIP-GFPislets by intravenous administration of alloxan (75 mg/kg), awell-characterized diabetogenic compound that is taken up by β-cells viathe glucose transporter 2 (26). This treatment rendered micehyperglycemic with a blood glucose concentration of 25.0±1.3 mmol/l(n=6) after 24 h. At this time-point, substantial loss of GFPfluorescence and structural changes in the reflection of the isletgrafts were observed (FIG. 4e-f ), indicating loss of β-cells.Administration of annexin V-APC (n=4) 24 h after the induction of celldeath resulted in strong labelling of islet grafts (FIG. 4 gh). Highmagnification imaging revealed that most annexin V-APC labelling wasfound in graft regions devoid of GFP fluorescence. Some annexin V-APCfluorescence was found on the surface of GFP-fluorescent β-cells,indicating labeling of cells undergoing apoptosis (FIG. 4i-l ). Weconclude that β-cell death can be imaged non-invasively andlongitudinally under in vivo conditions in islets engrafted in theanterior chamber of the eye.

Summary

We have now introduced a novel platform for non-invasive studies ofislet cell both physiology and pathophysiology in vivo. Employing theanterior chamber of the eye as an in vivo model for islet cell researchenables the continuous monitoring of morphology, vascularisation,innervation, cell death, and cell signalling. The use of this platformto study islet cell signal-transduction in vivo will help elucidatingthe effects of modulatory inputs from the hormonal and neuronal system,as well as from autocrine/paracrine signals of endocrine or vascularcells. Furthermore, it will serve as a novel approach for non-invasivein vivo studies of β-cell function and survival under healthy anddiabetic conditions. Noteworthy is, that this platform is not limited tostudies of pancreatic β-cell signal-transduction but can readily beextended to investigate numerous other cell types and organ tissues invivo. Hence, the anterior chamber of the eye can be used as a versatilenatural body window to clarify, for the first time, the integration ofcomplex signalling networks at the cellular level under in vivoconditions.

References for Example 1

-   1. Wajchenberg, B. L. Beta-Cell Failure in Diabetes and Preservation    by Clinical Treatment. Endocr Rev (2007).-   2. Berggren, P. O. & Leibiger, I. B. Novel aspects on    signal-transduction in the pancreatic beta-cell. Nutr Metab    Cardiovasc Dis 16 Suppl 1, S7-10 (2006).-   3. Vetterlein, F., Petho, A. & Schmidt, G. Morphometric    investigation of the microvascular system of pancreatic exocrine and    endocrine tissue in the rat. Microvasc Res 34, 231-8 (1987).-   4. Woods, S. C. & Porte, D., Jr. Neural control of the endocrine    pancreas. Physiol Rev 54, 596-619 (1974).-   5. Rahier, J., Goebbels, R. M. & Henquin, J. C. Cellular composition    of the human diabetic pancreas. Diabetologia 24, 366-71 (1983).-   6. Köhler, M. et al. Imaging of Pancreatic Beta-Cell    Signal-Transduction. Curr. Med. Chem.-Immun., Endoc.& Metab. Agents    4, 281-299 (2004).-   7. Speier, S. & Rupnik, M. A novel approach to in situ    characterization of pancreatic beta-cells. Pflugers Arch 446, 553-8    (2003).-   8. Shapiro, A. M. et al. Islet transplantation in seven patients    with type 1 diabetes mellitus using a glucocorticoid-free    immunosuppressive regimen. N Engl J Med 343, 230-8 (2000).-   9. Paty, B. W., Bonner-Weir, S., Laughlin, M. R., McEwan, A. J. &    Shapiro, A. M. Toward development of imaging modalities for islets    after transplantation: insights from the National Institutes of    Health Workshop on Beta Cell Imaging. Transplantation 77, 1133-7    (2004).-   10. Philipson, L. H. & Roe, M. W. Imaging. Curr. Med. Chem.-Immun.,    Endoc. & Metab. Agents 4, 333-337 (2004).-   11. Menger, M. D., Yamauchi, J. & Vollmar, B. Revascularization and    microcirculation of freely grafted islets of Langerhans. World J    Surg 25, 509-15 (2001).-   12. Porksen, N. et al. Coordinate pulsatile insulin secretion by    chronic intraportally transplanted islets in the isolated perfused    rat liver. J Clin Invest 94, 219-27 (1994).-   13. Meier, J. J. et al. Intrahepatic transplanted islets in humans    secrete insulin in a coordinate pulsatile manner directly into the    liver. Diabetes 55, 2324-32 (2006).-   14. Hultquist, G. T. The ultrastructure of pancreatic tissue from    duct-ligated rats implanted into anterior chamber of rat eyes. Ups J    Med Sci 77, 8-18 (1972).-   15. Niederkorn, J. Y. Immune privilege in the anterior chamber of    the eye. Crit Rev Immunol 22, 13-46 (2002).-   16. Adeghate, E. & Donath, T. Morphological findings in long-term    pancreatic tissue transplants in the anterior eye chamber of rats.    Pancreas 5, 298-305 (1990).-   17. Adeghate, E., Ponery, A. S., Ahmed, I. & Donath, T. Comparative    morphology and biochemistry of pancreatic tissue fragments    transplanted into the anterior eye chamber and subcutaneous regions    of the rat. Eur J Morphol 39, 257-68 (2001).-   18. Vetterlein, F., Petho, A. & Schmidt, G. Distribution of    capillary blood flow in rat kidney during postischemic renal    failure. Am J Physiol 251, H510-9 (1986).-   19. Menger, M. D., Vajkoczy, P., Leiderer, R., Jager, S. &    Messmer, K. Influence of experimental hyperglycemia on microvascular    blood perfusion of pancreatic islet isografts. J Clin Invest 90,    1361-9 (1992).-   20. Valdeolmillos, M., Santos, R. M., Contreras, D., Soria, B. &    Rosario, L. M. Glucose-induced oscillations of intracellular Ca2+    concentration resembling bursting electrical activity in single    mouse islets of Langerhans. FEBS Lett 259, 19-23 (1989).-   21. Mathis, D., Vence, L. & Benoist, C. beta-Cell death during    progression to diabetes. Nature 414, 792-8 (2001).-   22. Butler, A. E. et al. Beta-cell deficit and increased beta-cell    apoptosis in humans with type 2 diabetes. Diabetes 52, 102-10    (2003).-   23. Boersma, H. H. et al. Past, present, and future of annexin A5:    from protein discovery to clinical applications. J Nucl Med 46,    2035-50 (2005).-   24. Medarova, Z., Bonner-Weir, S., Lipes, M. & Moore, A. Imaging    beta-cell death with a near-infrared probe. Diabetes 54, 1780-8    (2005).-   25. Nyqvist, D., Köhler, M., Wahlstedt, H. & Berggren, P. O. Donor    islet endothelial cells participate in formation of functional    vessels within pancreatic islet grafts. Diabetes 54, 2287-93 (2005).-   26. Szkudelski, T. The mechanism of alloxan and streptozotocin    action in B cells of the rat pancreas. Physiol Res 50, 537-46    (2001).-   27. Köhler, M. et al. On-line monitoring of apoptosis in    insulin-secreting cells. Diabetes 52, 2943-50 (2003).-   28. Lipp, P. & Niggli, E. Ratiometric confocal Ca(2+)-measurements    with visible wavelength indicators in isolated cardiac myocytes.    Cell Calcium 14, 359-72 (1993).

Example 2

In this example, we provide a step-by-step protocol for non-invasivelongitudinal in vivo studies of cell biology at single-cell resolution,taking advantage of the cornea as a natural body window. For thispurpose, the tissue of interest is transplanted into the anteriorchamber of the eye and cell biological parameters are assessed by LSMthrough the cornea. The anterior chamber of the eye has been frequentlyused as a transplantation site to study a variety of tissues³⁻⁷. Whileoriginally the anterior chamber of the eye was selected as atransplantation site because of its properties as an immune privilegedsite⁸, most studies utilized the anterior chamber in a syngeneictransplantation setting because it is easily accessible and the corneaallows macroscopic observation of the engrafted tissue. Additionally,the iris, which forms the base of the anterior chamber, has one of thehighest concentrations of blood vessels and autonomic nerves in thebody, and thereby enables fast innervation⁹ and vascularization¹⁰ of thegraft. Up to date studies utilizing the anterior chamber as atransplantation site mainly employed macroscopic observations⁵ toinvestigate graft physiology. This restricts longitudinal studies toparameters observable at low resolution. In vivo electrophysiology⁷ aswell as histology³ and various other in vitro techniques after graftremoval enable assessment of morphology and cellular function, howeversetting an endpoint to the study and hereby preventing longitudinalmonitoring.

Materials Animals

-   -   NMRI mice (Charles River Laboratories, USA) or (Scanbur, Sweden)    -   Tie2-GFP mice [STOCK Tg(TIE2GFP)287Sato/J] (Jackson Laboratory,        USA)    -   Transgenic mice with fluorescent reporters expressed in        pancreatic beta-cells under the insulin promoter (RIP-GFP)¹¹.

Reagents

-   -   Sterile phosphate buffered saline (PBS)    -   Isoflurane (Isoflurane, Abott/Baxter, USA)    -   40% O₂ in 60% N₂ (AGA, Sweden)    -   Butrenorphine (Temgesic, Schering-Plough, USA)    -   Viscotears® (Novartis, Switzerland)    -   Alloxan (Sigma, USA)    -   Texas Red 70 kDa dextran (Invitrogen, USA)    -   Annexin V APC (Invitrogen, USA)    -   Hypnorm® (VetaPharm, UK)    -   Dormicum® (Roche, Switzerland)    -   Sterile water for injection (Braun, Germany)    -   Fluo-4 AM special packaging (Invitrogen, USA)    -   Fura-Red AM special packaging (Invitrogen, USA)    -   Pluronic® F-127 (Invitrogen, USA)    -   Glibenclamide (Sigma, USA)    -   Extracellular solution

Reagent Setup

Extracellular solution (in mM): 140 NaCl, 5 KCl, 2 NaHCO₃, 1 NaH₂PO₄,1.2 MgCl₂, 2.5 CaCl₂, 10 HEPES, 3 glucose (pH 7.4 with NaOH).

Equipment

-   -   27 G×¾″ needle (BD, USA)    -   Blunt 27 G cannula, custom made of a 27 G needle    -   0.5 ml threaded plunger Hamilton gastight syringe #1750        (Hamilton, USA)    -   polythene tubing 0.4 mm inner diameter (i.d.), 0.8 mm outer        diameter (o.d.) (Smiths Medical, UK)    -   polythene tubing 0.2 mm i.d., 0.8 mm o.d.    -   polythene tubing 0.9 mm i.d., 1.2 mm o.d.    -   tygon tubing 0.76 mm i.d., 0.86 mm wall thickness (Ismatec,        Switzerland)    -   1, 5 and 10 ml plastic syringes (BD, USA)    -   50 ml reagent tube (Eppendorf, Germany)    -   400 Anesthesia Unit (Univentor, Malta)    -   Exmire Microsyringe MS-GLLX00 10 ml (Hamilton, USA)    -   Stereomicroscope MZ FLIII (Leica, Germany)    -   Head holding adapter (SG-4N, Narishige, Japan)    -   UST-2 Solid Universal Joint (Narishige, Japan)    -   Dumont #5 Forceps (Fine Science Tools, USA)    -   Custom-made heating pad    -   DMLFSA upright microscope, equipped with a TCS-SP2-AOBS confocal        scanner (Leica, Germany)    -   Ti:Sapphire laser Tsunami (Spectra-Physics, USA)    -   2.5× and 5× objectives (Leica, Germany)    -   long distance water-dipping lenses (Leica HXC APO 10×0.3 W,        20×0.5 W, 40×0.8 W)    -   Micromanipulator 5171 (2) (Eppendorf, Germany)    -   Universal capillary holder (2) (Eppendorf, Germany)    -   Capillary grip head 1 (2) (Eppendorf, Germany)    -   Thin-wall borosilicate glass capillaries without filament        TW120-4 (WPI, USA)    -   DMZ universal puller (Zeitz Instrumente, Germany)    -   802 syringe pump (Univentor, Malta)    -   Leica Confocal Software (version 2.61) (Leica, Germany)    -   Volocity (Improvision, UK)    -   Matlab (The MathWorks, USA)    -   Wavelet filtering algorithm¹⁴ (Stockholm, Sweden)

Equipment Setup Confocal and Two-Photon Setup

For LSM we use a Leica TCS-SP2-AOBS confocal laser scanner equipped withArgon and HeNe lasers connected to a Leica DMLFSA microscope. Two-photonexcitation is achieved using a Ti:Sapphire laser (Tsunami;Spectra-Physics, USA) for ˜100 fs excitation at ˜82 MHz. The microscopestage is customized for the use of head holding adaptor and mouse.

Head Holding Adapter

To fix the head of the mouse for surgery and imaging, we employ a headholding adapter SG-4N-S manufactured by Narishige, Japan. Depending onthe type of anesthesia used the head holder is equipped with a gas mask(GM-4-S) or a nosepiece. The head holder is attached to a metal platewhich fits onto the customized stage of the microscope. The metal plateis covered by a heating pad. Body temperature is controlled via a rectalprobe which regulates the temperature of the heating pad.

Eye Stabilizer

For retraction of the eye lids and additional stabilization of the eyewe use a custom-made supporting device. Attach a #5 Dumont forceps to asmall metal bar and clamp the metal bar into a UST-2 Solid UniversalJoint. Fix the Universal Joint to the same metal plate as the headholder on level with the eye, so you can reach the eyes with the tips ofthe forceps. Cover the tips of the forceps with a piece of polyethylenetube, creating a loop between the tips. At the front part of the forcepsattach a screw to enable adjustment of the distance between the forcepstips.

Anterior Chamber Perfusion

Outflow: Connect a ˜35 cm piece of polythene tubing 0.9 mm i.d. and 1.2mm o.d. with one end to a capillary holder and connect the other end ofthe tubing to a 10 ml syringe without a plunger (open reservoir).Inflow: Connect a ˜35 cm piece of tygon tubing 0.76 mm i.d. and 0.86 mmwall thickness with one end to a capillary holder and connect the otherend of the tubing to a 1 ml syringe.

Image Processing

To denoise images captured with confocal LSM and TPLSM use waveletfiltering¹⁵. For analysis and image display use processing software e.g.Volocity and Leica confocal software.

Non-Limiting, Exemplary Procedure Transplantation of Pancreatic Isletsto the Anterior Chamber of the Eye

-   1 Isolate mouse pancreatic islets as previously described^(16,17).-    Optionally culture islets, depending on study parameters.-   2 Connect about 10 cm of 0.4 mm i.d. polythene tubing with a 27 G    needle to the 0.5 ml Hamilton syringe and insert the blunt 27 G    cannula at the other end of the tubing.-   3 Transfer 30-40 islets from the culture medium to a dish with    sterile PBS and center the islets as compact as possible in the    middle of the dish.-   4 Aspirate the islets into the blunt 27 G cannula and the connected    polythene tubing. The islets are preferably aspirated in a minimal    volume (for example, 20 μl or less) to facilitate injection into the    anterior chamber.-    Aspirating the islets into too big a volume may lead to    difficulties during the injection process by exposing the eye to    unnecessary high intraocular pressure and may result in reflux of    islets out of the anterior chamber after removing the cannula.-   5 Carry out the transplantation of islets to the anterior chamber of    the eye according to option (A) or (B).

(A) Transplantation of Islets Using a Head Holder.

-   -   i Put a piece of cotton wool in a 50 ml reagent tube and drop        about 1 ml Isoflurane onto the wool. Stun the mouse by holding        it for a few seconds into the reagent tube.    -   ii Place the mouse in the head holder under a stereomicroscope        and fix the head with the eye selected for transplantation        facing upwards.    -   iii Anesthetize the mouse using Isoflurane, 2-2.5% in 40% O₂ and        60% N₂. Isoflurane levels are carefully controlled to ensure a        proper state of anesthesia. Vacuum is applied in the area of        anesthesia to protect the operator.    -   iv Inject butrenorphine (0.05 mg/kg) subcutaneously to relieve        post-operative pain.    -   v Carefully pull back the eyelids and gently place the        polyethylene tubing loop of the eye stabilizer below the        corneoscleral junction.    -    Care is taken when placing the stabilizing forceps to avoid        disrupting blood circulation in the eye.    -   vi Connect a 27 G needle to a 1 ml syringe to ease handling. Use        the 27 G needle to puncture the cornea close to the sclera while        taking care not to damage the iris and to avoid bleeding.    -   vii Gently insert the blunt cannula into the anterior chamber of        the eye through the hole made with the needle. Slowly inject the        islets into the anterior chamber. After injection, carefully        withdraw the cannula.    -   viii Leave the mouse in the head holder before awakening for        additional 10-15 min. Remove the mouse from the head holder,        turn off the Isoflurane and observe the mouse during awakening.    -   ix Put a drop of Viscotears on the eye to prevent desiccation.    -    A break is made until the in vivo imaging. The duration depends        on study parameters.

(B) Transplantation of Islets without Using the Head Holder.

-   -   i Prepare a small gas mask from a 5 ml plastic syringe by        removing the piston and cutting the syringe ˜1 cm above the        bottom. Connect the tubing of the anesthetic pump to the needle        fitting.    -   ii Stun the mouse by holding it for a few seconds into the 50 ml        reagent tube with cotton and about 1 ml Isoflurane.    -   iii Place the mouse under a stereomicroscope on a heating pad        with the eye selected for transplantation facing upwards. Put        the nose of the mouse in the prepared gas mask.    -   iv Anesthetize the mouse using Isoflurane, 2-2.5% in 40% O₂ and        60% N₂. Isoflurane levels are carefully controlled to ensure a        proper state of anesthesia. Vacuum is applied in the area of        anesthesia to protect the operator.    -   v Inject butrenorphine (0.05 mg/kg) subcutaneously to relieve        post-operative pain.    -   vi Retract the skin around the eye to visualize the        corneoscleral junction of the eye and gently fix the position of        the head, without interrupting the breathing or blood        circulation of the mouse.    -   vii Continue as described above under (A) from point vi to ix.

Imaging of Islets Engrafted in the Anterior Chamber of the Eye

-   6 Transplant islets according to steps 1-5. Choose donor and    recipient mice depending on the aim of the study.-   7 Stun the recipient mouse by short exposure to Isoflurane.-   8 Place the mouse in the head holder and fix the head with the eye    containing the transplanted islets facing upwards.-   9 Anesthetize the mouse using Isoflurane, 2-2.5% in 40% O₂ and 60%    N₂.-    Isoflurane levels are carefully controlled to ensure a proper state    of anesthesia. Vacuum is applied in the area of anesthesia to    protect the operator.-   10 Carefully pull back the eyelids and gently place the polyethylene    tubing loop of the eye stabilizer below the corneoscleral junction.-    Care is taken when placing the stabilizing forceps to not disrupt    blood circulation in the eye.-   11 Place the head holder together with the mouse under an upright    microscope equipped for confocal and two-photon LSM.-   12 To get an overview use low magnification objectives (2.5 and 5×).    For high resolution LSM utilize water immersion dipping objectives    (10, 20, and 40×) with a long-working distance using filtered saline    or Viscotears as immersion liquid between the lens and the cornea.    -   Apply the minimum required laser-power and scan-time necessary        for visualization to avoid photodamage and bleaching; for        example, below 75 mW, and from 800 Hz and above.-   13 Image the biological parameter of interest:    -   (A) Imaging of graft morphology        -   i To visualize cell specific morphology, islets of            transgenic mice expressing a fluorescent protein in            beta-cells (e.g. RIP-GFP) can be used for transplantation.            Excite GFP fluorescence with a 488 nm laser and detect            emission between 495 and 530 nm. Islet morphology can also            be imaged by detection of a reflection image. Choose a laser            (e.g. 633 nm) and set the AOBS control to optimize            reflection detection. Collect emission between ±4 nm of the            laser wavelength.    -   (B) Imaging of vascularization in the iris and the engrafted        islets    -   Visualize vascularization either by imaging endothelial cells or        the blood vessel lumen.        -   i To image endothelial cells utilize Tie2-GFP mice for            transplantation.        -   ii Excite GFP fluorescence with a 488 nm laser and detect            emission between 495 and 530 nm.        -   iii For imaging of the blood vessel lumen inject 0.1 ml of            10 mg/ml of a fluorescently labeled dextran (70 kDa)            intravenously into the tail vein.        -   iv Following injection of a fluorescently labeled dextran,            image the engrafted islets using appropriate settings for            the chosen dextran.        -    For simultaneous imaging of beta-cells and vessels            transplant islets of RIP-GFP mice and inject Texas-Red            conjugated dextran (70 kDa). Excite Texas Red and GFP with a            two-photon laser at 890 nm and collect emission light onto            non-descanned detectors using a dichroic mirror (RSP 560)            and emission filters (BP 525/50 and BP 640/20).    -   (C) Imaging of beta-cell death        -   i Induce beta-cell death in the engrafted islets by            intravenous injection of alloxan (75 mg/kg body weight).        -    Wait for 24 h for alloxan to induce beta-cell death.        -   ii Measure blood glucose levels 24 h after the            administration of alloxan to confirm that the mouse has been            rendered hyperglycemic.        -   iii Inject 0.1 ml of annexin V-APC intravenously via the            tail vein.        -    Wait for 4-6 h for annexin V APC to label apoptotic and            dead cells.        -   iv Image beta-cell death in the engrafted islets between 4-6            h following the administration of annexin V-APC, using            appropriate settings for APC fluorescence. Excite APC at 633            nm with collection of emission light between 645-680 nm.    -   (D) Imaging of cytoplasmic free Ca²⁺ concentration after loading        the graft with Ca²⁺ indicators via perfusion of the anterior        chamber of the eye.        -   Anterior chamber perfusion is modified from ref.¹⁸.            -   i Pull pipettes for the anterior chamber of the eye                perfusion from glass capillaries using a regular pulling                program for patch pipettes. Break the pipette to a tip                diameter of 30-40 μm. Bevel the tips at an angle of 35                degrees to a final diameter of 70-90 μm. Pipettes for                the outflow should be slightly bigger (˜90 μm) than                pipettes for the inflow (˜70 μm).            -   ii Fill the syringes, tubing and capillary holders with                filtered extracellular solution and place the pipettes                in the capillary grip head of the capillary holder.            -   iii Anesthetize the mouse by an intraperitoneal                injection of 100 μl/10 g bodyweight of a Hypnorm/sterile                water/Dormicum mix (1:2:1). Anesthesia will set in                within 1-2 min. Prolong anesthesia by injections of 50                μl/10 g bodyweights of a Hypnorm/sterile water mix (1:3)                after 30 and 60 min. If necessary further prolong                anesthesia after 90 min by an injection of 50 μl/10 g                bodyweight of the initial Hypnorm/sterile water/Dormicum                mix (1:2:1).            -    Care is taken in choosing an anesthetic for functional                studies on islet cells, as several compounds have been                reported to exert an effect on blood glucose levels and                insulin secretion^(19,20). Isoflurane has been shown to                inhibit glucose stimulated insulin release by a direct                mechanism on islet cells and therefore is not suitable                for functional studies²¹. A mix of Hypnorm/Dormicum does                not seem to interfere with the measurements of changes                in cytoplasmic free Ca²⁺ concentration.            -   iv Put the mouse in the head holder and fix the head                with the eye containing the transplanted islets facing                upwards.            -   v Carefully pull back the eyelids and gently place the                polyethylene tubing loop of the eye stabilizer below the                corneoscleral junction and place holder and mouse under                the upright microscope.            -    Care is taken when placing the stabilizing forceps not                to disrupt blood circulation in the eye.            -   vi Hang the open reservoir of the outflow at a height of                ˜21 cm above the eye to ensure a constant intraocular                pressure of ˜15 mm Hg. Place the capillary holder of the                outflow in the micromanipulator.            -   vii Employ a 2.5× objective while inserting the outflow                pipette into the anterior chamber with the                micromanipulator to observe the entire eye.            -    Penetrate the anterior chamber by moving the pipette                fast through the cornea at a shallow angle. Be careful                not to unnecessarily scratch the cornea or to damage the                iris.            -   viii Aspirate ˜130 μl of the Fluo-4/Fura-Red mix (1:1,                500 μM each) into the inflow capillary and fix the                capillary holder onto the micromanipulator. Make sure                that there is enough dye-free extracellular solution in                the syringe for wash out. Place the 1 ml syringe of the                inflow into the syringe pump.            -   ix Insert the inflow pipette into the anterior chamber                opposite to the outflow pipette. Penetrate the cornea in                the same way as in step vii.            -   x Initially, exchange the aqueous humor with the                perfusate fast (˜10 μl in 30 s). Observe functionality                of the perfusion.            -   xi Continuously perfuse the anterior chamber of the eye                at a rate of ˜3 μl/min for ˜40 min.            -   xii After loading, wash out the dye in the anterior                chamber by perfusing the anterior chamber at a fast rate                (˜10 μl/min).            -    During perfusion steps, control the perfusion and the                eye. Pay attention that the eye is not swelling due to a                blocked outflow.            -   xiii After wash out of the dye switch off the perfusion.                Do not remove the pipettes.            -   xiv For imaging of changes in cytoplasmic free Ca²⁺                concentration switch to a higher magnification water                immersion dipping objective (10, 20 or 40×) and apply                Viscotears as immersion liquid.            -   xv Simultaneously image Fluo-4 and Fura-Red to enable                ratiometric measurements of changes in cytoplasmic free                Ca²⁺ concentration. Excite Fluo-4 and Fura-Red at 488 nm                and collect emission light for Fluo-4 between 495-535 nm                and for Fura-Red between 600-700 nm.            -    Apply the minimum required laser-power and scan-time                necessary for visualization to avoid photodamage and                bleaching; for example, below 75 mW, and from 800 Hz and                above            -   xvi Start acquiring a time series of the Fluo-4 and                Fura-Red fluorescence in the cells of interest.            -   xvii Acquire a baseline of unstimulated fluorescence                levels and stimulate systemic insulin release by                injecting glibenclamide (1 mg/kg) intravenously via the                tail vein. Changes in cytoplasmic free Ca²⁺                concentration in beta-cells within the islet graft                should be observed within seconds after injection.            -   xviii After imaging remove the pipettes carefully from                the eye.            -   xix To relieve post-operative pain inject the mouse                subcutaneously with butrenorphine (0.05 mg/kg).            -   xx Place the mouse in a warm environment (˜30° C.) until                it wakes up. After Hypnorm/Dormicum anesthesia this can                take several hours.

Timing

Isolation of islets (step 1): ˜4 hTransplantation of islets to the anterior chamber (steps 2-5): ˜25min/mouseImaging of graft morphology (step 13 A): ˜1 h/mouseImaging of graft vascularization (steps 13 B, i-ii or iii-iv): ˜1h/mouseImaging of beta-cell death (steps 13 C i-ii): ˜24 h; (step 13 C iii-iv):˜5 hImaging of cytoplasmic free Ca²⁺ concentration after loading the graftwith Ca²⁺ indicators via perfusion of the anterior chamber of the eye(step 13 D i): ˜1.5 h/4-6 pipettes; (steps 13 D ii-xx): ˜2 h/mouse

Anticipated Results

The here introduced platform enables longitudinal assessment of severalbiological parameters in vivo without the need of invasive surgicalprocedures to access the tissue of interest. Following transplantationof pancreatic islets of Langerhans to the anterior chamber of the eye,the current protocol easily enables detection of islet graft morphologyby reflection or fluorescence imaging. Morphological characterization ofa pancreatic islet graft by imaging reflection and GFP was carried outin which. islets of mice expressing GFP under the insulin promoter(green beta-cells) were transplanted to mice expressing GFP under theTie2 promoter (green endothelial cells). GFP was excited with 488 nm at35% laser power and emission measured between 495-530 nm. Reflection wasimaged by exciting with 633 nm at 35% laser power and measuring emissionbetween 632-639 nm.

Furthermore, vascularization and cell death can be followedlongitudinally by systemic injections of fluorescent dyes. Blood vesselswere visualized by an intravenous injection of a 70 kDa Texas Redlabeled dextran. GFP and Texas Red were excited with a two-photon laserat 890 nm at minimal necessary laser power required and emissioncollected onto non-descanned detectors using a dichroic mirror (RSP 560)and emission filters (BP 525/50 and BP 640/20). For imaging of celldeath, beta-cell death was induced by intravenous injection of alloxan.Apoptotic and dead cells were visualized by intravenous injection ofannexin V-APC. Reflection was imaged by exciting with 543 nm andemission measured between 539-547 nm at 35% laser power. GFP was excitedat 488 nm and emission measured between 495-530 nm at 35% laser power.APC was excited at 633 nm with collection of emission light between645-680 nm at 75% laser power.

Additionally, islet-cells can be loaded repetitively with Ca²⁺indicators, by perfusion of the anterior chamber, to measuresystemically induced changes in cytoplasmic free Ca²⁺ concentration. Forloading with calcium indicators, the anterior chamber was perfused withFluo-4 and Fura-Red. Fluo-4 and Fura-Red were excited with 488 nm at 25%laser power and emission measured for Fluo-4 between 495-535 nm and forFura-Red between 600-700 nm. Reflection was imaged by exciting with 543nm and emission measured between 539-547 nm at 15% laser power.

Extending the current protocol by the use of various transgenic mice andindicators will enable the observation of numerous additional biologicalparameters. Thereby, this platform will help to investigate cell biologyof complex systems under physiological as well as pathophysiologicalconditions.

Example 2 References

-   1. Koo, V., Hamilton, P. W. & Williamson, K. Non-invasive in vivo    imaging in small animal research. Cell Oncol 28, 127-139 (2006).-   2. Handbook of biological confocal microscopy, Edn. 3 (Pawley, J.    B.) (Springer, New York, N.Y., 2005).-   3. Adeghate, E., Ponery, A. S., Ahmed, I. & Donath, T. Comparative    morphology and biochemistry of pancreatic tissue fragments    transplanted into the anterior eye chamber and subcutaneous regions    of the rat. European journal of morphology 39, 257-268 (2001).-   4. Katoh, N., et al. Target-specific innervation by autonomic and    sensory nerve fibers in hairy fetal skin transplanted into the    anterior eye chamber of adult rat. Cell and tissue research 266,    259-263 (1991).-   5. Olson, L. & Seiger, A. Beating intraocular hearts:    light-controlled rate by autonomic innervation from host iris.    Journal of neurobiology 7, 193-203 (1976).-   6. Wu, W., Scott, D. E. & Reiter, R. J. Transplantation of the    mammalian pineal gland: studies of survival, revascularization,    reinnervation, and recovery of function. Experimental neurology 122,    88-99 (1993).-   7. Hoffer, B., Seiger, A., Ljungberg, T. & Olson, L.    Electrophysiological and cytological studies of brain homografts in    the anterior chamber of the eye: maturation of cerebellar cortex in    oculo. Brain research 79, 165-184 (1974).-   8. Niederkorn, J. Y. Immune privilege in the anterior chamber of the    eye. Critical reviews in immunology 22, 13-46 (2002).-   9. Adeghate, E. Pancreatic tissue grafts are reinnervated by    neuro-peptidergic and cholinergic nerves within five days of    transplantation. Transplant immunology 10, 73-80 (2002).-   10. Adeghate, E. Host-graft circulation and vascular morphology in    pancreatic tissue transplants in rats. The Anatomical record 251,    448-459 (1998).-   12. Zhuravleva, Z. N., Bragin, A. G. & Vinogradova, O. S.    Organization of the nervous tissue (hippocampus and septum)    developing in the anterior eye chamber. I. General characteristic    and non-neural elements. Journal fur Hirnforschung 25, 313-330    (1984).-   13. Adeghate, E. & Donath, T. Distribution of neuropeptide Y and    vasoactive intestinal polypeptide immunoreactive nerves in normal    and transplanted pancreatic tissue. Peptides 11, 1087-1092 (1990).-   14. Boutet de Monvel, J., Le Calvez, S. & Ulfendahl, M. Image    restoration for confocal microscopy: improving the limits of    deconvolution, with application to the visualization of the    mammalian hearing organ. Biophysical journal 80, 2455-2470 (2001).-   15. Kohler, M., et al. On-line monitoring of apoptosis in    insulin-secreting cells. Diabetes 52, 2943-2950 (2003).-   16. Berney, T., et al. Endotoxin-mediated delayed islet graft    function is associated with increased intra-islet cytokine    production and islet cell apoptosis. Transplantation 71, 125-132    (2001).-   17. Nyqvist, D., Köhler, M., Wahlstedt, H. & Berggren, P. O. Donor    islet endothelial cells participate in formation of functional    vessels within pancreatic islet grafts. Diabetes 54, 2287-2293    (2005).-   18. Bernd, A. S., Aihara, M., Lindsey, J. D. & Weinreb, R. N.    Influence of molecular weight on intracameral dextran movement to    the posterior segment of the mouse eye. Investigative ophthalmology    & visual science 45, 480-484 (2004).-   19. Aynsley-Green, A., Biebuyck, J. F. & Alberti, K. G. Anaesthesia    and insulin secretion: the effects of diethyl ether, halothane,    pentobarbitone sodium and ketamine hydrochloride on intravenous    glucose tolerance and insulin secretion in the rat. Diabetologia 9,    274-281 (1973).-   20. Brown, E. T., Umino, Y., Loi, T., Solessio, E. & Barlow, R.    Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.    Visual neuroscience 22, 615-618 (2005).-   21. Desborough, J. P., Jones, P. M., Persaud, S. J., Landon, M. J. &    Howell, S. L. Isoflurane inhibits insulin secretion from isolated    rat pancreatic islets of Langerhans. British journal of anaesthesia    71, 873-876 (1993).    troubleshooting

TABLE 1 PROBLEM POSSIBLE REASON SOLUTION Movement of the graft Movementof the head Fix the clamping of the head during imaging (Step 12).because the head holder is holder not properly fixed. Movement of theeye during Adjust the eye stabilizer. normal breathing. carefully.Gasping of the mouse. Make sure that the airflow of the anesthesia unitand isofluorane levels are set properly. No fluorescence can be Tailvein injection failed. Repeat tail vein injection. detected in the bloodvessels Excitation and emission Adjust excitation and (Step 13 B ii).settings are wrong. emission settings. Blood circulation to the eyeAdjust the position of the eye is disrupted due to false stabilizer.setting of eye stabilizer. Perfusion does not work Air bubbles block theMake sure that there are no (Step 13 D x). pipettes, capillary holder orair bubbles in the tubing, tubing. capillary holders or capillaries,prior to the experiment. Dirt blocks the pipette tips. Be sure to cleanthe pipettes after beveling, right before the experiment.

I claim:
 1. A method for treating a subject with diabetes, comprisingtransplanting into an eye of a subject with diabetes an amount effectiveof insulin-producing cells to promote insulin production in the subject.2. The method of claim 1, wherein the insulin-producing cells arepresent in an isolated pancreatic islet.
 3. The method of claim 1,wherein the insulin-producing cells comprise isolated pancreatic βcells.
 4. The method of claim 1, wherein the transplantation into theeye involves transplantation onto the iris of the eye.
 5. The method ofclaim 1, wherein the treating comprises restoring normoglycemia.
 6. Themethod of claim 2, wherein the transplantation into the eye involvestransplantation onto the iris of the eye.
 7. The method of claim 3,wherein the transplantation into the eye involves transplantation ontothe iris of the eye.
 8. The method of claim 2, wherein the treatingcomprises restoring normoglycemia.
 9. The method of claim 3, wherein thetreating comprises restoring normoglycemia.
 10. The method of claim 4,wherein the treating comprises restoring normoglycemia.
 11. The methodof claim 6, wherein the treating comprises restoring normoglycemia. 12.The method of claim 7, wherein the treating comprises restoringnormoglycemia.